Bumetanide prevents transient decreases in muscle force in murine hypokalemic periodic paralysis

Abstract

Objective: To test the hypothesis that inhibition of the Na-K-2Cl transporter with bumetanide will reduce the susceptibility to decreases in muscle force in a mouse model of hypokalemic periodic paralysis (HypoPP).

Methods: In vitro contraction tests were performed on soleus muscle isolated from mice with knock-in missense mutations that result in HypoPP (sodium channel NaV1.4-R669H) or hyperkalemic periodic paralysis (HyperPP; sodium channel NaV1.4-M1592V).

Results: Bumetanide prevented the development of weakness in 2 mM K(+) and also restored force during an established attack of HypoPP. Stimulation of the Na-K-2Cl transporter via induction of hyperosmolality exacerbated the weakness seen in low K(+) and was also prevented by bumetanide. Bumetanide was more efficacious than acetazolamide for preventing weakness in low K(+) conditions. Decreases in force in HyperPP muscle exposed to 10 mM K(+) were not prevented by treatment with bumetanide.

Conclusions: The Na-K-2Cl inhibitor bumetanide was highly effective in preventing attacks of weakness in the NaV1.4-R669H mouse model of HypoPP and should be considered for management of patients with HypoPP due to sodium channel mutations. Dehydration may aggravate HypoPP by stimulating the Na-K-2Cl transporter.

Figures

Figure 1. Bumetanide prevents reduction in force in low K+ conditions

(A) Force recorded during a tetanic contraction of the soleus muscle from a heterozygous R669H+/m mouse in 4.75 mM K+ bath solution (left), 20 minutes after switching to 2 mM K+ (middle), and 12 minutes after application of bumetanide (BMT) (right). Stimulus (blue) is shown below each force response. (B) Peak tetanic force was measured every 2 minutes and normalized to the amplitude in the 4.75 mM K+ bath solution. Exposure to 2 mM K+ elicited 90% decrease in force for R669Hm/m muscle (black squares, n = 8), a 60% decrease in R669H+/m (gray squares, n = 7), and only a 30% decrease in WT (open squares, n = 8). The partial recovery in force between 50 and 60 minutes for R669Hm/m is due to spontaneous oscillations with cyclical recovery and weakness. Addition of 75 μM BMT produced a full recovery of tetanic force. (C) Application of BMT at the onset of a 2 mM K+ exposure prevents decreases in force. Washout of BMT caused a large reduction in force for R669Hm/m muscle.

Figure 2. Hyperosmolality-induced decreases in force that were aggravated by low K+ conditions and prevented by bumetanide

(A) Soleus from R669H+/m mice (n = 6) was more susceptible to reductions in force due to hyperosmolar challenge as compared to WT (n = 6). A concomitant reduction in K+ levels from 4.75 to 2 mM resulted in a complete loss of force for R669H+/m soleus. (B) Pretreatment with bumetanide (BMT) (75 μM) prevented decreases in force.

Figure 3. Beneficial effect of bumetanide was Cl− dependent

Tetanic contractions were measured in Cl−-free conditions. Peak forces recorded from the left and right soleus in separate tissue baths are superimposed. Both muscles were exposed to 2 mM K+ starting at 10 minutes, and only one preparation had bumetanide (BMT) (75 μM) applied at 40 minutes (red squares). (A) For soleus from a heterozygous R669H+/m mouse, the low-K+ challenge induced only a small decrease in force. Application of bumetanide did not produce recovery. Myotonia was evident from the slowed relaxation after a contraction (inset, contractions recorded before and after replacement of Cl− by methanesulfonate). (B) Low K+ triggered a large decrease in force for the soleus muscle from a homozygous R669Hm/m mouse. Spontaneous fluctuations in peak force occurred during continuous exposure to 2 mM K+, but the addition of BMT (40 minutes, red symbols) did not prevent subsequent transient decreases.

Figure 4. Bumetanide was superior to acetazolamide in preventing decreases in force under low K+ conditions

Sequential 2-mM K+ challenges were applied to soleus from heterozygous R669H+/m female (left) or male (right) mice. In each trial, a pair of muscles from the same animal was tested in separate tissue baths. The control had no drug applied (gray) while the other muscle was pretreated with acetazolamide (ACTZ) (100 μM) (red) in the first low-K+ challenge and bumetanide (BMT) (0.5 μM) before the second challenge. The modest improvement with ACTZ was not significantly different from untreated (p > 0.3, n = 6). BMT prevented decreases in force in females, and substantially reduced such decreases in males (p < 0.01, n = 3).

Figure 5. Bumetanide was not effective in preventing reductions in force in hyperkalemic periodic paralysis

Peak tetanic force in soleus muscle of M1592V hyperkalemic periodic paralysis mice decreased by 35%, when challenged with 10 mM K+ (n = 6). Application of 75 μM bumetanide (BMT) did not produce a recovery, whereas return to 4.75 mM K+ resulted in complete recovery.